Biomass produced as part of services in treating industrial and/or municipal wastewater can be purposefully enriched with PHA accumulating bacteria (PAB). The PAB-rich biomass exhibits a significant potential to accumulate PHA when fed with waste organic streams containing readily biodegradable chemical oxygen demand (RBCOD). RBCOD is typically comprised of but is not limited to volatile fatty acids (VFAs). When PAB are fed with RBCOD, PHA may be made to accumulate in the biomass to significant levels of the final biomass dry weight. The PAB-rich biomass is mixed with an RBCOD-rich wastewater under aerobic or anoxic conditions so as to maximize the PHA yield. Typically, the final biomass dry weight is well in excess of 40% as PHA where one kilogram of active biomass dry weight can typically be made to accumulate in excess of two thirds of a kilogram PHA dry weight. This PHA content is distributed in the PAB in the biomass as small intracellular granules imbedded in the cytoplasm and these granules can range up to about 0.5 μm in diameter. The inclusions are surrounded by their own phospholipid membrane which contains proteins for the inclusion synthesis. PHA can be recovered from the biomass as a polymer with purity in excess of 95% and even up to 99%. The recovered polymer will be referred to as recovered PHA-resin. The objective in the development of this disclosed invention has been to:    1. establish a viable process for recovery of PHA resin from biomass,    2. maximize yield of PHA mass recovered from the biomass in the process,    3. minimize the potential for uncontrolled decrease in PHA molecular weight during solvent extraction, and    4. facilitate a means to extract a PHA resin exhibiting high thermal stability.
The PHA produced by this biomass is typically, but not exclusively, poly(3-hydroxybutyric acid) and/or a co-polymer of 3-hydroxybutyric and 3-hydroxyvaleric acids. The PHA that can be recovered from this biomass is a biodegradable polyester or biopolymer exhibiting physical properties similar to plastics like polypropylene (PP) and polyethylene (PE). PHAs can be compounded into plastics or further converted into central platform chemicals. However, unlike PP and PE, PHA is completely biodegradable. The invention represents a sub-component of the overall biorefinery concept for a wastewater treatment process which can comprise but is not limited to the following elements (FIG. 1):                I. A unit process designed to convert some or all of the organic material in the influent to an RBCOD rich wastewater and to remove all the RBCOD from the wastewater while producing a biomass with an extant potential for significant accumulation of PHAs.        II. A means for controlled retention and metered export of the produced biomass for purposes of stable wastewater treatment process operation and PHA production.        III. A unit process for accumulating PHA to significant levels in the produced biomass by using a RBCOD rich feed derived from the same wastewater or other on- or off-site waste organic sources.        IV. A unit process to make the PHA-in-biomass thermally stable and resistant to decomposition due to elevated temperatures or chemical interactions while ensuring parallel benefit in, for example, energy production from any non-PHA material removed from this biomass.        V. A unit process to extract a PHA resin with thermal stability.The present invention is focused on step IV, namely, preparing the PHA in the biomass for recovery and as a necessary step towards eventual purification (V) from this PAB-rich biomass after accumulation. The invention provides practical solutions for meeting product quality objectives in PHA recovery and satisfying parallel wastewater treatment and waste residual handling performance and savings objectives as explained further below.        
Recovery of PHA from the PHA-rich biomass is the problem of separation of the granules from the other non-PHA cellular material (NPCM) that contains both organic and inorganic fractions. The quality for PHA resin recovered from biomass for use as an ingredient to the formulation of plastics may be assessed in terms of its: (1) purity, (2) average molecular weight and its distribution, (3) thermal stability, (4) chemical stability, and (5) co-polymer microstructure and composition. Purity refers to the remaining biomass NPCM and perhaps also other chemicals or elements introduced or carried over during the purification process.
Average molecular weight reflects the average size of the polymer chain lengths. In most cases PHA is a polymer with a relatively broad molecular weight distribution. Mn is the number average molar mass and it is defined as:
      M    n    =                    ∑                                      ⁢                        N          i                ⁢                  M          i                                    ∑                                      ⁢              N        i            where Ni is the number of molecules with molar mass Mi. The weight average molar mass, Mw, is defined as:
      M    w    =                    ∑                                      ⁢                        N          i                ⁢                  M          i          2                                    ∑                                      ⁢                        N          i                ⁢                  M          i                    The polydispersity index (PDI), a measure of the molecular weight distribution, is defined as:
  PDI  =            M      w              M      n      Mw is always larger than Mn so the PDI will always be greater than 1. PDI for PHA-resin is typically around 2 and Mw can range from 10,000 to 3,000,000 Da. Molecular weight distribution can be influenced by the method of accumulating PHA in the biomass, the method for recovering the PHA resin and the method of further processing the resin into end-user products.
Thermal stability refers to the resistance of the polymer to decomposition as a function of temperature and time in a specified atmosphere. The atmosphere can be inert (such as nitrogen) or reactive (such as air or oxygen). Thermal stability can be assessed in terms of a characteristic decomposition temperature of sample volatilization or weight loss. Practically, the stability of the polymer at or slightly above its processing temperature is also relevant. Thus, thermal stability is also assessed by the kinetics of degradation in processing, which is to say molecular weight decrease and/or dynamic viscosity decrease, in air or an inert atmosphere such as nitrogen gas, and at relevant processing temperatures. Chemical stability here refers to the tendency for polymer degradation, in contact with liquids (solvents, non-solvents, aqueous, non-aqueous or mixtures thereof). Chemical reactions may initialize due to temperature with transport and diffusion of compounds or reactive groups which will be more mobile with the polymer in contact with liquid. Chemical reaction products in the liquid environment may further promote the degradation process.
Mixed cultures can be made to produce homopolymers and co-polymers of PHA and the type and distribution of the monomers in the co-polymer influence processing characteristics and the final material properties. For example, feeding the biomass with butyric or acetic acid represents RBCOD for the accumulation of poly(3-hydroxybutric acid) (PHB). Feeding biomass an RBCOD mixture of acetic and propionic acids can promote production of copolymers of 3-hydroxybutric acid and 3-hydroxyvaleric acids (PHBV).
Impurities in the PHA resin may be organic and/or inorganic. While a high purity is desirable, some impurities may be acceptable due to the fact that different impurities exhibit differing effects on the polymer properties or processability. Generally we have been striving for PHA recovery to absolute purity in excess of 95% and ideally in excess of 99%. The impurities are typically related to organic and inorganic components found in biomass NPCM. The organic impurities can include protein, carbohydrate, and lipid residuals from the biomass. The inorganic impurities can include cations such as calcium, magnesium, sodium and corresponding anions such as phosphate, sulphate, and chloride. The PHA impurity may also be due to moisture retention which is a reflection of the presence of undesirable organic or inorganic impurities. Organic impurities can produce undesirable off-colouring and pungent odours in PHA resin processing into plastics and products. Organic impurities are also implicated in the polymer chemical stability. Inorganic impurities can severely reduce the resin thermal stability. PHA in dried PHA-rich biomass after an accumulation process may typically range from 40 to 60% of the dry mass.
The chemical and thermal stabilities of PHA in pure and mixed culture biomass after accumulation are typically poor and temperatures in excess of 100° C. cannot be used in the biomass processing or PHA recovery if excessive molecular weight loss is to be avoided.
Given that up to 60 percent of dried PHA-rich biomass may be NPCM, one may concurrently need to resolve the fate of this non-PHA fraction along with efficient PHA recovery. Eventual disposal of excess biomass created from biological treatment of wastewater has become a global problem. Significant efforts in research and development have been devoted to technology development that maximizes the recovery of energy and resources from wasted biomass and minimizes the material requiring secure disposal.
NPCM fate is a constraint for PHA recovery within the context of excess biomass used to treat wastewater. NPCM residuals may be compatible with and ideally further improve the state-of-the-art in environmental protection when it comes to solids handling at wastewater treatment facilities. Much technology and development expense is being devoted to correct for deficiency and problems created by current process solutions for sludge handling at wastewater treatment plants. Ideally the approach for PHA recovery should not only generate potential in value added biopolymer production but also opportunity in greater and more effective control in overall residual solids management.
In the conversion of biopolymer PHA from a purified resin into plastic, additives may be combined. These components are combined at or slightly above the processing temperature of the resin and the mixture is extruded and formed into plastic pellets. In a melting cycle the final amount of PHA in the material may be reduced due to mixing additives with the melt. Usually it is intended to compound the PHA into plastic pellets and these pellets then become a raw feed used in the production of end-user products that again requires heating and forming the plastic. Thus, the PHA resin generally survives at least two heating cycles before becoming an end-user product. For every heating cycle the PHA in the plastic will reduce in average molecular weight. The plastic material properties are influenced directly and indirectly by the resin molecular weight. For example, viscosity of the melt decreases with molecular weight and the processability of the plastic is sensitive to the melt viscosity. Too high or too low a viscosity can be equally undesirable. Notwithstanding, predictable behaviour of the polymer in the melt is preferred and so a polymer of consistent thermal and chemical stabilities is of general benefit. The mechanical properties depend on the final molecular weight of the processed polymer and molecular weight decrease can mean a negative influence on the product mechanical properties.
The level of average molecular weight decrease depends on factors including the PHA thermal and chemical stabilities, time in the melt, temperatures, screw speeds, and shear forces applied for processing. Degradation of the polymer in the melt is also strongly influenced by chemical impurities or additives that can add to or detract from the polymer stability for a given processing temperature. The non-PHA fraction in the end material may comprise of the original impurities present in the extracted resin. The non-PHA fraction will also consist of the added organic and inorganic compounds.
The objectives of the added compounds can be summarized as follows:    1. Thermal stabilizing agents help to reduce unwanted polymer degradation in the melt that impair the processability of the end product material properties. They can include both inorganic and organic additives and introduce costs to the plastic production from PHA. Examples are zinc oxides, zinc stearate, magnesium stearate, calcium stearate, barium stearate and phosphonic acids.    2. Nucleating agents increase the onset and rate of crystallization of the polymer from the melt and fast crystallization is of practical importance for industrial plastic processing and service life of the final product. The final PHA morphology is influenced which in turn affects the material properties. Examples are boron nitride, talc, palmitic acid, oleic acid, linoleic acid, saccharin, ammonium chloride, stearic acid salts and phosphonic acids.    3. Plasticizing compounds lower the glass transition of the polymer making it softer, decreasing brittleness and thereby increasing elongation and impact strength. These additives play a role for short chain length PHAs like PHB and PHBV that are brittle. Examples are acetyl trialkyl citrates, triacetin, phthalate esters, maleate, sebacate, adipate, PHA oligomers, diols and triols.    4. Colouring agents impart specific colouring to the plastic. Colour requirements are very demanding and so it is desirable that any colouration tendency from the resin due to processing is consistent and can be compensated for.    5. Composites involve an additive that serves a functional role of filler and augments the material mechanical properties. For example, the biomass NPCM can be considered to be a filler and the resultant composite has been used as a biodegradable container for tree seedlings where mechanical property demands are not onerous. Natural fibres can also be used as a composite with PHA where the polymer provides the matrix for the fibre network. Such fibre reinforced PHA can exhibit the strength of PHA with impact resistance far in excess of PHA alone. Fillers may also be used to reduce the cost of the plastic by reducing the PHA content so long as the composite material properties meet the application demands.
The recovered PHA from biomass has been observed to exhibit poor thermal and chemical stability. The native resin available on the market today degrades rapidly in the melt. Molecular weight loss in processing can only be permitted up to the point where melt viscosity becomes too low or final product quality is adversely impacted. However, such molecular weight loss margins may not be practically feasible to obtain within processing times. Thermal stability of recovered PHA resin can be improved by further purification to remove the implicated impurities or by adding stabilization agents. This will add complexities and expenses. In addition, some stabilization agents may create a liability due to potential for environmental problems in the life cycle of PHA-based plastic products.
NPCM from biomass can be removed to upgrade the PHA content of the biomass recovered from an accumulation process. NPCM can be removed using treatment strategies involving mechanical, chemical, enzyme, and thermal means or combinations thereof. Optimal NPCM removal conditions involve treatment duration, chemical/enzyme concentration, energy input, and temperature. Notwithstanding the potential to improve the PHA content of the biomass to well above 70%, such treatment may promote some degradation of the PHA in the biomass. Lysis of cells and release of PHA granules into the matrix can further complicate the separation of the PHA inclusions from other cellular debris and reduce product yield. Significant solubilisation of the biomass creates a downstream wastewater treatment problem due to organic carbon, nitrogen and phosphorus release into solution.
Even if the PHA content can be upgraded, conditioning of the biomass creates a parallel waste management liability due to concomitant release of carbon, nitrogen and phosphorus. This liability can be converted into an asset in situations where the carbon can be used to produce biogas and the nutrients can be recycled for biological treatment of nutrient deficient industrial wastewaters. However, yield in polymer recovery may be reduced due to impaired capture of suspended solids following cell disruption leading to mixtures of cellular debris and PHA granules.
Even if PHA content can be upgraded by biomass conditioning, production of a PHA resin may ultimately always require some form of solvent extraction. The PHA is separated from NPCM by dissolving the PHA in a solvent. If temperatures above 100° C. are to be avoided due to poor thermal stability of the PHA in the biomass then chlorinated solvents such as chloroform and dichloromethane may be necessary. When these solvents are used to extract the PHA, a non-solvent such as water or methanol is used to precipitate the PHA from the solvent after filtering the non-dissolved NPCM. Large volumes of hazardous waste are generated due to the combination of a chlorinated solvent and the co-solvent used.
When it comes to PHA, if the biomass has been conditioned to at least the extent of improved thermal and chemical stabilities of the PHA in the matrix, then requisite temperatures in the process of PHA recovery can range between 100 and 160° C. with little molecular weight loss. In this temperature range a number of poor solvents can extract the PHA from the NPCM. These are solvents that do not dissolve PHA under 100° C. but are good solvents to extract PHA above 100° C. Examples of such solvents are acetone, butanol, propanol, ethanol, methanol, and 1,2 propylene carbonate, among others. However, the stability of PHA dissolved in these solvents is also solvent type and isomer dependent. Some PHAs can be extracted at lower temperatures so that requisite solvent and extraction temperature is also PHA dependent. PHB presents one worst case scenario for solubility and solvent extraction. Notwithstanding potential for exposure to elevated temperatures required for PHA-resin solvent extraction from biomass, biomass drying in general before solvent extraction may be more effectively accomplished at temperatures well above 100° C. For example, dual belt low temperature dryer may mean exposure of the biomass to temperatures between 140 and 180° C.
The NPCM residual after extraction still contains most of the organic carbon, nitrogen and phosphorous of the original biomass. This extraction residual is hygienic and a suitable feedstock for biological and thermo-chemical technologies yielding platform chemicals and/or energy. The NPCM or subsequent residuals after chemical/energy extraction can also be used directly in product formulations intended to supply nutrients and minerals for agriculture. Therefore the method of PHA recovery may also be seen as a parallel method for capture of other value added residuals while avoiding waste management problems as compared to current state-of-the-art in sludge management.
Development efforts for a PHA recovery solution from PHA-rich biomass have been with the following central objectives in mind:    1. Limit the need for chemical additions so as to reduce process complexity and costs associated with waste biomass processing,    2. Enable higher temperature biomass drying before resin extraction,    3. Enable higher temperature resin solvent extraction from the biomass with non-chlorinated solvents by ensuring the PHA in the biomass is thermally and chemically stable before solvent extraction,    4. Facilitate the production of a PHA of high thermal stability in order to significantly reduce the need for stabilizing agents in plastic formulation, and    5. Capture a source of NPCM residual that can be readily managed and exploited for its mineral, organic, and calorific contents.